1. Field of the Invention
This invention relates to immunoassay compositions and methods for determining the presence of amphetamines in a sample suspected of containing amphetamine and/or methamphetamine. In particular, the immunoassay compositions of this invention for determining the presence amphetamines in a sample suspected of containing amphetamine and/or methamphetamine are comprised of two conjugates containing functionally similar labels, one bound to an amphetamine ligand analog and the other bound to a methamphetamine ligand analog. These immunoassay compositions may also include an antibody to amphetamine and an antibody to methamphetamine wherein at least one of the antibodies is a monoclonal antibody. The compositions of this invention are particularly useful for assaying for amphetamines even in the case where the monoclonal antibody is moderately cross-reactive with the conjugate which is bound more strongly by the other antibody.
2. Related Art
A wide variety of patents and literature references disclose different immunoassay techniques. The following list is merely illustrative of some of these techniques which can find application in this invention. The following is a list of Unites States patents and a general statement of the type of label involved:
U.S. Pat. Nos. 3,646,346, Radioactive Label; 3,654,090, 3,791,932 and 3,817,837, (Rubonstein) et al.) Enzyme Labels; 3,996,345; Flurorescer-Quencher Labels; 4,062,733, Radioactive Label; 4,067,959, Fluorescer or Enzyme Labels; and 4,160,645, Non-Enzymatic Catalyst Label.
Typically, these immunoassays will employ an antibody whose structure will recognize (have a binding affinity for) the analyte specific for that antibody. The immunoassay is conducted with a signal producing system which produces a detectible change in signal upon binding of the analyte to the antibody. Accordingly, when testing for an analyte in a sample, a detectible change in signal from that produced with a negative sample or calibrator is taken as a positive result for the presence of that analyte in the sample.
However, there is a problem when such techniques are employed to assay for amphetamines in a sample suspected of containing amphetamine and/or methamphetamine. The problem arises because these assays employ a single antiserum or antibody which can recognize both amphetamine and methamphetamine. In order for this antibody to recognize both amphetamine and methamphetamine, it is necessary for it (a) to be capable of recognizing a particular spatial and polar organization common to amphetamine and methamphetamine; and (b) to lack specific recognition of those structural features of amphetamine and methamphetamine that are different. Because such an antibody recognizes structural features that are common to both of these compounds but lacks specific recognition of the structural features that are different, it is able to recognize both compounds and the assay will produce a positive result for a sample containing amphetamine and/or methamphetamine. However, all antibodies that recognize both compounds have been found to recognize molecules other than amphetamine and methamphetamine that share some but not all of the common spatial and polar features of amphetamine and methamphetamine. Such antibodies may produce false positive results in the assay of certain samples, i.e., a positive result for amphetamines in the absence of amphetamine and methamphetamine. In point of fact, commercial immunoassays for amphetamines which utilize a single antibody either are sensitive to only amphetamine or methamphetamine or recognize certain prescription and OTC drugs and normal metabolic products which are structurally similar to amphetamine and methamphetamine including drugs such as phenylpropanolamine, pseudoephedrine, phentermine, mephentermine, etc., and the metobolic product tyramine. As a result, test samples containing one or more of these drugs or metabolic products can produce false positives for amphetamines.
This problem of false positives arising from testing for amphetamines using a single antibody is particularly troublesome because in actual practice, every positive test result requires a follow-up test to confirm the presence of amphetamines. Such follow-up tests inherently add additional laboratory time and expense to the immunoassay particularly if a substantial portion of these positive results are false positives. Although in principle separate tests could be run each using an antibody specific for one drug, this too increases the number of tests that must be run. Accordingly, it would be particularly desirable to develop an immunoassay which could test for amphetamines while at the same time result in substantially reduced occurrences of false positives arising from compounds which are structurally related to amphetamines.
This would not be a difficult problem if antibodies could be prepared that were completely specific for amphetamine and would not bind to a ligand analog of methamphetamine that was bound to a detectible label, and antibodies specific for methamphetamine were available that would not bind to a ligand analog of amphetamine that was bound to a detectible label. If such antibodies could be made, the components of two specific assays could be combined to provide a single assay for amphetamines. However, such antibodies are not available and the binding of one antibody to the label conjugate of a ligand analog of the other drug might be expected to prevent the construction of a useful assay.
What we have found and what the instant invention is directed toward is an immunoassay composition which can assay for the presence of amphetamines in a sample suspected of containing amphetamine and/or methamphetamine while producing substantially fewer false positives wherein two antibodies are used together with conjugates of a ligand analog of each of the drugs with a label.
U.S. Pat. No. 4,329,281 discloses 4-[4-[2-(aminopropyl)]phenyl]butanoic acid and its N-methyl derivative as useful in preparing immunogens which can be respectively employed in the elicitation of polyclonal antibodies selective to amphetamine and methamphetamine. These antibodies are then used in immunoassays.
U.S. Pat. No. 4,041,076 discloses amphetamine and methamphetamine derivatives substituted at the para phenyl position with --O(CH.sub.2).sub.n CO.sub.2 H wherein n is 1 to 3. These derivatives are useful in preparing immunogens which can be employed to elicit polyclonal antibodies selective to amphetamine and methamphetamine. These antibodies are then used in immunoassays.
U.S. Pat. No. 4,067,774 discloses amphetamines bound to enzymes via a --OZC(O)--group on the phenyl ring wherein Z is hydrocarbylene of from 1 to 7 carbon atoms. These enzyme linked compounds are used in immunoassays.
U.S. Pat. Nos. 3,996,344 and 4,016,146 discloses the preparation of amphetamine and methamphetamine analogs which are useful in preparing immunogens which can be employed to elicit polyclonal antibodies selective to amphetamine and methamphetamine. The compounds so disclosed are substituted on the phenyl group with HOOCCH.sub.2 CH.sub.2 C(O)--.
Lastly, N-carboxymethyl amphetamine has been employed in preparing immunogens which can be employed to elicit a polyclonal antibody which recognizes amphetamine and methamphetamine.